Dendritic spines are small neuronal protrusions that form the postsynaptic half of the majority of excitatory synapses in the adult CMS. Prior to the formation of dendritic spines during development, however, dendrites are covered with motile, actin-rich protrusions called filopodia. As synaptogenesis proceeds over the first two postnatal weeks, filopodia are believed to mature directly into spines but the mechanisms underlying this transformation are not known. At the same developmental ages at which dendritic filopodia are being replaced by dendritic spines, the number and type of ionotropic glutamate receptors (NMDA and AMPA receptors) expressed at excitatory synapses is changing. Many immature synapses express only NMDARs (i.e. AMPAR "silent synapses"). Because NMDAR activation leads to AMPAR insertion in long- term potentiation in the adult CMS, and also initiates changes in the shape and number of dendritic spines, it is likely that NMDAR activation plays a critical role in the formation and maturation of excitatory synapses during development. I propose to test three hypotheses concerning the critical role of glutamate in the genesis of mature axo-spinous synapses. AIM 1: NMDAR activation promotes the outgrowth of filopodia from the dendritic shaft. AIM 2: Filopodial NMDAR activation triggers their transformation into dendritic spines. AIM 3: Filopodia lack functional AMPARs until NMDAR activation induces their insertion. To test these hypotheses, I will combine laser microphotolysis of caged glutamate at individual dendritic filopodia with simultaneous live cell fluorescence imaging of the stimulated process and electrophysiological recordings of the glutamate receptor-mediated responses in the stimulated cell. Experiments will be performed on CA1 pyramidal neurons in organotypic hippocampal slice cultures made from rats and GFP- expressing mice at postnatal day 1-4. The combination of these techniques provides me with the unique ability to answer fundamental questions of how glutamate drives the process of synaptic maturation. Understanding the mechanisms underlying spine formation is a critical first step in understanding the etiology of disorders, such as Fragile X syndrome and other forms of mental retardation, that are characterized by immature and/or abnormal axo-spinous synapses. RELEVANCE: We will elucidate how excitatory contacts between brain cells develop and mature normally. This will provide not only a better understanding of the defective formation of these contacts in many forms of mental retardation, but also the insight needed to develop new therapeutic and preventative strategies.